dry food product containing live probiotic

ABSTRACT

The disclosure relates to a probiotic delivery system that can be consumed as a snack-food or added to a food product. In particular, the disclosure describes a crisp and tasty treat that comprises viable probiotic microorganisms preserved in a vacuum dried matrix of sugars, proteins, and polysaccharides. The probiotic remain viable within the treat for a longer time without the need for additional moisture barrier coating. The probiotic also remain viable in the animal gastrointestinal tract.

BACKGROUND OF THE DISCLOSURE

1. Field of the Invention

The disclosure relates generally to the fields of probiotics and food.

2. Related Art

The activity and long term stability of many biological materials, suchas proteins, enzymes and microbial cells may be affected by a number ofenvironmental factors; for example, temperature, pH, the presence ofwater and oxygen or oxidizing or reducing agents. Generally, biologicalmaterials must be dried before or during mixing with other foodstuffingredients. The drying process can often result in a significant lossin activity from mechanical, chemical, and osmotic stresses induced bythe drying process. Loss of activity occurs at many distinct stages,including drying during initial manufacturing, feed preparation (hightemperature and high pressure), transportation and long term storage(temperature and humid exposure), and after consumption and passage inthe gastrointestinal (GI) track (exposure to low pH, proteolytic enzymesand bile salts). Manufacturing food or feedstuffs with live cellorganisms or probiotics is in particular challenging, because theprobiotics are very sensitive to the drying process and to temperatureand moisture conditions of the foodstuff. Another concern is theprobiotic resistance in the acid environment in the stomach and itssuccessful colonization of the intestine.

Probiotic microorganisms (probiotics) are living microorganisms, whichupon ingestion in certain numbers, exert health benefits beyond basicnutrition. The beneficial effects that probiotics may induce arenumerous. Few examples are; the reduction of lactose intolerance, theinhibition of pathogenic bacteria and parasites, the reduction ofdiarrhea, activity against Helicobacter pylori, the prevention of coloncancer, the improvement or prevention of constipation, the in situproduction of vitamins, the modulation of blood lipids, and themodulation of host immune functions. In domesticated and aquatic animalsthey also can improve growth, survival and stress resistance associatedwith diseases and unfavorable culture conditions. Therefore, there isconsiderable interest in including probiotics into human foodstuffs andinto animal feed.

Many probiotics exhibit their beneficial effect mainly when they arealive. Hence, they need to survive the manufacturing process and shelflife of the food, and upon consumption of the food where they need topass through the gastro-intestinal tract before reaching their place ofcolonization. Although many commercial probiotic products are availablefor animal and human consumptions, most of them lost their viabilityduring the manufacture process, transport, storage and in the animal GItract (see the viability studies of several probiotic products by(Hughes and Hillier 1990; Shah 2000). To compensate for such loss, anexcessive quantity of probiotics is included in the product inanticipation that a portion will survive and reach their target. Inaddition to questionable shelf-life viability for these products, suchpractices are certainly not cost-effective. Alternatively, the probioticmicroorganisms can be encapsulated in protective microenvironments.Generally, current microencapsulation and enteric coating techniquesinvolve applying a film forming substance, usually by spraying liquidscontaining sugars or proteins onto the dry probiotics (Ko and Ping WO02/058735). However, coating the microencapsulated probiotics withmoisture protecting layers is an expensive process, and generallyseveral layers must be added, to avoid water entering the microcapsules.In addition, it is extremely difficult to remove the added liquid in thecoating substance without a corresponding decrease in shelf life.

Various protective agents have been used in the art, with varyingdegrees of success. These include proteins, certain polymers, skim milk,glycerol, polysaccharides, oligosaccharides and disaccharides.Disaccharides, such as sucrose and trehalose, are particularlyattractive cryoprotectants because they are actually help plants andmicrobial cells to remain in a state of suspended animation duringperiods of drought. Trehalose has been shown to be an effectiveprotectant for a variety of biological materials, both in ambientair-drying and freeze-drying (Crowe et al. 1998). However, there aresome drawbacks associated with the use of sugars as the solecryoprotectant. For example, large amounts of sugars (often greater than60% by weight) must be used to preserve the biological materials duringthe drying process. This is costly. More serious problems associatedwith the use of sugars include their readiness to form crystals when thematerial is dried below its freezing point, and the low glass transitiontemperature which causes instability of the preserved biologicalmaterials at high temperatures, and/or in humid environments. Further,high concentration of sugars reduces the solubility of other solutes inthe system and at the same time renders the system extremely difficultto dry.

Accordingly, it has been proposed to dry sugar-based probiotic systemsby foam formation in a very thin layer (Bronshtein WO2005117962), or touse combinations of sugars with a polymeric gelling agent, such asalginate, chitosan, carboxymethylcellulose or carboxyethylcellulose.Cavadini et al. (EP 0 862 863) provide a cereal product comprising agelatinized starch matrix including a coating or a filling. Theprobiotic is included with the coating. According to that process,spray-dried probiotics are mixed with a carrier substrate, which may bewater, fat or a protein digest. The mixture is then sprayed onto thecereal product and the whole product is dried again. Re-hydrating of thealready dried bacteria and the additional coating/drying process iscostly and damaging to the bacteria.

Kenneth and Liegh (U.S. Pat. No. 6,900,173) describe the manufacturingof multivitamin protein and probiotic bar for promoting an anabolicstate in a person. The dried probiotic bacteria are blended in sugarsyrup and several other constituents, and the resultant mixture is thenextruded and cut into bars. However, the document does not disclose anyprocess or composition that will improve viability or long-termstability of probiotics in the nutritional bars and there is noindication that the bacteria even survive the process.

Ubbink et al. (US 2005/0153018) disclose the preservation of lactic acidbacteria in moist food. The spray-dried bacteria are added to acomposition comprising fats, fermented milk powder and saccharides. Thatcomposition is then used as the filling of a confectionary product. Thesubject matter described in that document avoids the detrimental effectsof water by embedding the probiotics in fat or oil rich matrix. However,fat based coating and preserving materials do not withstand long termexposure to humid conditions.

Giffard and Kendall (US 2005/0079244) disclose a foodstuff in the formof a dried or semi-moist ready-to-eat kibble or powder mix, whichcontains a combination of a probiotic, prebiotic and a coating ofcolostrum. Prior to mixing in the food stuff, the probiotic is coated orencapsulated in a polysaccharide, fat, starch, protein or in a sugarmatrix using standard encapsulation techniques. Similar to the abovedisclosure, the negative effects of water were avoided by embedding theprobiotics in a matrix rich in fat or oil.

Farber and Farber (WO 03/088755) describe an oral delivery system forfunctional ingredients uniformly dispersed in a matrix. The matrixcomponents include a sugar, a carbohydrate, a hydrocolloid a polyhydricalcohol and a source of mono- or divalent cations. The delivery systemis extruded or molded into a final shape with a moisture content ofbetween 15% and 30% by weight. This type of matrix provides very littleprotection to the probiotics mostly under refrigerated conditions. Nodescription or direction was provided as to how probiotic bacteria arestabilized during manufacturing or for prolonged storage at roomtemperatures.

Porubcan (US 2004/0175389) discloses a formulation for protectingprobiotic bacteria during passage through the stomach, whilst permittingtheir release in the intestine. The formulation has also a low wateractivity and correspondingly long shelf life. The capsule includes awater-free mixture of probiotic bacteria with monovalent alginate salts,and an enteric coating (e.g., gelatin or cellulose encapsulation). Uponcontact with acidic environment, the outer shell of the capsule turnedinto a gel, which provides a protecting barrier against proton influxinto the capsule core. However, this composition is only useful forlarge particles such as tablets and capsules subjected to storageconditions of very low water activity and further require storage innitrogen-flushed or vacuum-sealed containers. McGrath and Mchale (EP1382241) describe a method of delivering a microorganism to an animal.The micro-organism is suspended in a matrix of cross-linked alginate andcryopreservant (trehalose or lactose, or a combination of both). Thematrix is then freeze or vacuum dried to form dry beads containing liveprobiotics with a shelf-life stability up to 6 months but only underrefrigerated conditions. Here again, no description or direction wasprovided as to how probiotic bacteria are stabilized duringmanufacturing or for prolonged storage at room temperatures and highhumidity conditions.

None of the above compositions provide a mixture that can effectivelyprotect the probiotic in both drying processes and long-term storage atelevated temperatures and varying degrees of humidity. Therefore, thereis an urgent need for such a composition that can effectively protectthe probiotic bacteria during manufacturing, long-term storage atelevated temperatures and humidity and during gastrointestinal passage.There is a need also for a drying process that is cost-effective andcapable of entrapping and stabilizing probiotics in the protectivemixture with minimal viability loss at the end of the entire operation.There is a need for a protective mixture that provides protection in theanimal stomach while allowing the release of the probiotic along theintestinal tract. There is also a need for a protective mixture thatcontains only approved ingredients generally regarded as safe (GRAS),and is less costly than those presently being used.

The subject matter described herein overcomes these needs and provides acomposition and process for producing a composition that providesprobiotic bacteria that are stable for long periods of time even atelevated temperatures and varying degrees of humidity.

It is, in particular, a purpose of the present disclosure to describeviable probiotic cultures that are substantially stable at roomtemperature and high humidity conditions thereby obviate the need forrefrigeration or storage under vacuum or oxygen free environment.

BRIEF SUMMARY OF THE DISCLOSURE

It was unexpectedly found that probiotic bacteria are protected for anextended period of time in high temperature and humid conditions whenpreserved in a certain protective mixture. Additional qualities of theprotecting mixture are, a fast and cost effective drying process andgastric protection. The mixture comprises: (a) at least one sugarcompound, where the total amount of sugar compound in the mixture isfrom about 10% to about 60% by weight of the mixture (b) proteins, wherethe total amount of proteins in the medium is from about 2% to about 20%by weight of the mixture and (c) polysaccharides, where the total amountof polysaccharides in the medium is from about 0.5% to about 5% byweight of the mixture. This aqueous protective mixture can be used in amultiplicity of preservation processes, including freezing,freeze-drying, spray-drying, vacuum-drying, or ambient air-drying, toprovide a stable and preserved composition of probiotics. The probioticsubstance is stable for extended periods of time at superambienttemperatures and/or relative humidity. Further, the aqueous protectivemixture containing the probiotics can be molded into a desirable shapeor form and vacuum-dried to produce a crisp and tasty probiotic treatthat can be added to food stuff or consumed on its own by humans oranimals.

Therefore, the present disclosure also describes a method for preparinga preserved probiotic substances containing the above-noted protectivemixture.

Preferably, the probiotic substance is provided in a dry form that issubstantially free of water. The probiotic substance maybe freeze-dried,vacuum dried or air dried, or otherwise dried by methods known in theart. Accordingly, the probiotic substance preferably comprises aprotective mixture capable of maintaining the viability of the probioticmicro-organisms for extended periods of time in ambient temperature andhumidity conditions.

Preferably, the sugar in the protective mixture is a disaccharide, mostpreferably trehalose or sucrose or lactose or a combination thereof. Theprotective mixture preferably comprises trehalose at 20% w/v to 60% w/vtrehalose, preferably 20%, 30% or 40%.

Preferably, the protein in the protective mixture of the subject matterdescribed herein is egg albumen or soy protein isolate or hydrolysateand a mixture thereof. The protective mixture preferably comprisesproteins at 2% w/v to 20% w/v proteins, preferably 5%, 10% or 20%.

Preferably, the polysaccharides in the protective mixture describedherein can form a firm gel or viscous solution with the otheringredients in the mixture, most preferably a combination of alginateswith different viscosities, agarose, pectin or chitosan. The protectivemixture preferably comprises alginates at 0.5% w/v to 10% w/v alginates,preferably 1%, 2% or 4%.

In accordance with the subject matter described herein, there isprovided the use of the probiotic substance described herein for themanufacture of a probiotic product or a probiotic food or feed productfor the consumption by humans, domestic animals, aquatic animals andpets.

Remarkably, it was found that by adding a mixture containing 30%trehalose (w/v), 10% soy protein isolate (w/v) and 2% sodium alginate toa probiotic bacteria concentrate, forming viscous solution or hydrogeland vacuum drying it at temperature above the freezing point of themixture an excellent process recovery, prolonged stability over storagetime in ambient conditions and gastric protection are obtained.

Optionally, the probiotic substance may be coated with a moisturebarrier component. In principle, any food-grade substance having waterrepelling or impermeable properties may be selected. Suitable moisturebarriers may be, for example, a mixture of oil based substances.

Consequently, in a first aspect, the subject matter described hereinincludes a probiotic product comprising a dry micro-matrix particle ofthe above mixture, wherein the micro-matrix particle comprises viablemicroorganisms and has a size between 10 and 2000 microns.

In a second aspect, the subject matter described herein includes a foodproduct containing probiotic flakes or treats, wherein the flakes ortreats are the probiotic substance described herein, characterized inthat the flake or treat has a desirable shape and size between 2-50millimeters.

In a third aspect, the subject matter described herein includes aprocess for obtaining micro-matrix particles, to supplement a foodproduct with viable micro-organisms. The process comprises the steps ofmixing micro-organisms concentrate and further protective components,forming viscous solution or hydrogel, drying the mixture byfreeze-drying, spray-drying, vacuum-drying, or ambient air-drying, and,if necessary, grinding the dry material to obtain micro-matrix particlescomprising a size between 50-2000 microns.

In a fourth aspect, the subject matter described herein includes aprocess for obtaining flakes or treats as supplement or stand-alone foodproduct with viable microorganisms. The process comprises the steps ofmixing micro-organisms concentrate and further protective components,forming a hydrogel in a desirable shape and size and drying byvacuum-drying, to obtain crispy and tasty flakes or treats comprising asize between 2-50 millimeters.

One major advantage of the subject matter described herein is that itprovides a significant improvement over other drying methods of sugarbased substances and production methods of stable probioticmicro-organisms in semi-dry and/or humid particulate foodstuffs.

Another advantage of the subject matter described herein is that thedrying process is easy to upscale and straightforward with no need ofadditional coating or several drying stages.

Yet another advantage of the subject matter described herein is that theprobiotic substance provides gastric protection and a release mechanismof the probiotics along the intestinal tract at their site of action.

Yet another advantage of the subject matter described herein is that itprovides a suitable delivery vehicle for further functional ingredients,in particular non-digestible sugars, natural proteins and prebioticfibers, which in turn may improve the physico-chemical characteristicsof the probiotic substance, as described herein.

BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a table that shows drying loss after vacuum drying of L.rhamnosus probiotic in the MicroMatrix product described in Example 1.

FIG. 2 is a graph that illustrates storage stability (recovered cfu/g{colony-forming units per gram} versus storage time) of dry L. rhamnosusprobiotic substance stored at 40° C. and 33% relative humidity.

FIG. 3 is a bar chart that illustrates gastric stability (recoveredcfu/g) of probiotic substances incubated in simulated gastric juice(pH=1.2) at 37° C. for 2 hours.

FIG. 4 is an image of examples of probiotic “chips” produced asdescribed herein.

DETAILED DESCRIPTION

The disclosure relates to food products including a probiotic component.

The present disclosure relates to a food or feed product comprisingviable microorganisms, a mixture that protects probiotics against hightemperature, humidity and low pH, the use of the probiotic substance ina food product and a process for obtaining a probiotic substance tosupplement food or feed products. The disclosure further relates to afood product comprising the probiotic substance.

The subject matter described herein relates generally to a compositionfor preserving probiotic microorganisms, and to the production anddrying methods of the substance. More specifically, the subject matterdescribed herein includes a dry probiotic substance with long-term shelflife under high temperature and humid conditions.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The term “food product” is intended to encompass any consumable matterof either plant or animal origin or of synthetic sources that contain abody of nutrients such as a carbohydrate, protein, fat vitamin, mineral,etc. The product is intended for the consumption by humans or byanimals, such as domesticated animals, for example cattle, horses, pigs,sheep, goats, and the like. Pets such as dogs, cats, rabbits, guineapigs, mice, rats, birds (for example chickens or parrots), reptiles andfish (for example salmon, tilapia or goldfish) and crustaceans (forexample shrimp). Preferably, the subject matter described hereinincludes standard food products pelleted feeds, and pet food (forexample a snack bar, crunchy treat, cereal bar, snack, biscuit, petchew, pet food, and pelleted or flaked feed for aquatic animals).

The word “probiotic” is intended to refer to any consumablemicroorganism owing to any beneficial effect it may have on itsconsumer.

The term “probiotic substance” is a dry consumable substance in anyshape or form that contains probiotics. More specifically, a probioticsubstance comprises live probiotics embedded in a matrix of sugars,proteins and polysaccharides. Hence, it may be a food product on itsown.

The term “micro-matrix” particles may assume any dry powder form of theprobiotic substance. The micro-matrix particles may serve as a carrierfor the probiotics and comprise a size from 10 micron up to 2000 micron.

The term “flake or treat” is not intended to refer to specific form orshape of the probiotic substance. A flake or treat may assume any formobtained by molding shaping or slicing a hydrogel. For example, a flakeor treat may have the form of a sphere, cube, pyramid, tablet, cereal orany complex three-dimensional form that comprise a size of at least 2millimeters. For example, if the treats are used as a probiotic deliverysystem for pet-food, they may have the form of bones, rods, rings orother kibble forms.

The word “hydrogel” may refer to any moist food-grade substance that hasthe property of a solid or viscous gel. A hydrogel, either anionic orcationic, can be formed by one or more hydrophilic polymers,polysaccharides, gums, resins, or hydrolyzed proteins, either alone orin combination, in which the microorganisms are disposed. Preferably,the hydrogel compounds include agarose, alginate, chitosan or any othercompound, which preferably can present characteristics of a solid gel.

DETAILED DESCRIPTION

In order to prepare the probiotic substance as described herein, asingle or a mixture of several micro-organisms may be selected. As aprobiotic micro-organism, any micro-organism may be selected.Preferably, a micro-organism exerting beneficial effects on health andwelfare on humans or animals is used.

Examples of suitable probiotic micro-organisms include yeasts such asSaccharomyces cereviseae, molds such as Aspergillus, Rhizopus, Mucor,and Penicillium, bacteria such as the genera Bifidobacterium,Clostridium, Bacillus and Lactobacillus. Specific examples of suitableprobiotic micro-organisms are: Aspergillus niger, A. oryzae, Bacilluscoagulans, B. lentus, B, licheniformis, B, mesentericus, B. pumilus, B,subtilis, B. natto, Bifidobacterium adolescentis, B. animalis, B. breve,B. bifidum, B. infantis, B, lactis, B. longum, B. pseudolongum, B.thermophilum, Candida pintolepesii, Clostridium butyricum, Enterococcuscremoris, E. diacetylactis, Efaecium, E. intermedins, E. lactis, E.muntdi, E. thermophilus, Lactobacillus acidophilus, L. alimentarius, L.amylovorus, L. crispatus, L. brevis, L, case L, curvatus, L.cellobiosus, L. delbrueckii ss, bulgaricus, Lfarciminis, L. fermentum,L. gasseri, L. helveticus, L. lactis, L. plantarum, L. johnsonii, L.reuteri, L. rhamnosus, L. sakei, and L. salivarius.

The probiotic micro-organisms are preferably mixed in a concentrated wetpaste form or frozen paste form (for example a probiotic paste of >10%solids) with the other protective substances. Microorganisms may also bemixed, directly after fermentation, with the protective componentsdescribed herein followed by hydrogel formation and a drying processthereafter. For example, probiotic micro-organisms are mixed with theprotective materials such as a saccharide, for example trehalose,sucrose, lactose or maltodextrin, a protein, for example egg albumen,soy protein isolate or hydrolysate either alone or in combination and apolysaccharide, for example, agarose, alginate or chitosan either aloneor in combination. A hydrogel is then formed in a desired shape and sizeor sliced after hardening the gel according to established proceduresknown to persons skilled in the art. If micro-matrix particles arerequired, then the hydrogel can be sliced or extruded and then driedusing a variety of drying techniques, for example fluidized bed drying,freeze drying, air drying, convention oven drying or another adequatedrying process. The dry probiotic substance is then ground and sieved topreferred sizes. If flakes or treats are required, then the molded orotherwise pre-shaped or sliced hydrogel is preferably dried in a vacuumdrier or freeze drier at a temperature above the freezing point of thehydrogel. The pre-shaped dried flake or treat is then ready forpackaging alone or in combination with other food products.

Preferably, the probiotic substance comprises significant amounts of theprotective composition, in which the micro-organisms are embedded.Preferably, the probiotic substance comprises, in percent by weight oftotal dry matter, 1-50%, preferably 5-25%, more preferably 10-20% ofprobiotic microorganisms in the protective composition.

In one embodiment, the probiotic substance described herein comprises10⁶ to 10¹² viable micro-organisms (cfu) per gram dry weight.Preferably, it comprises 10⁷ to 10¹¹ cfu per gram dry weight.

In another embodiment, the dried probiotic substance is characterized bya water activity below 0.2. Preferably, the water activity is below 0.1,for example, the water activity is in the range of 0.01 to 0.09.

The probiotic substance in accordance with a preferred embodimentcomprises of sugar, proteins and polysaccharides. Wherein the protein isselected from natural proteins including albumen, arginine/lysinepolypeptide, collagen and hydrolyzed collagen, gelatin and hydrolyzedgelatin, glycoproteins, milk protein, casein, whey protein, serumalbumin, meat, fish, seafood, poultry, egg proteins, silk, soybean,corn, peanut, cottonseed, sunflower, pea, wheat protein, wheat germprotein, gluten-protein, zein and any isolate or hydrolyzed of anyvegetable protein, and the like.

Preferably, the polysaccharide components of the probiotic substance maybe selected in a way that a formation of a solid gel is possible.Generally, this may be achieved by cross linking the polysaccharide (forexample by mixing divalent cations with alginate or by cooling the gel(for example agarose).

Additional functional ingredients may be selected to provide furtherbenefits to the probiotic substance described herein. For examplefructo-oligosaccharides (FOS) and polyfructoses, for example, inulin,pectin, 6-glucans, resistant starches, for example high amylose starch,dextrans, acacia gum, guar and locust bean gum, agar, carrageenans,xanthan and maltodextrins, and mixtures thereof. Additional functionalingredients may also comprise trace elements, minerals, vitamins,antioxidants, sterols, antioxidants and/or other functional molecules.However, the effect of any additional components on the protectivecharacters of the probiotic substance should be evaluated first.Examples of vitamins and/or antioxidants are carotenoids, such aslycopene, beta-carotene, lutein, xanthophylls, vitamin A, tocopherols,vitamin C, and mixtures thereof.

Since one of the objectives of the compositions and processes describedherein is to add the probiotic substance to a food product, it is anadvantage that the probiotic substance may be shaped like the desiredfood product. For example, if the probiotic substance is to be added toa pet food, the probiotic substance may be shaped like a pellet, kibbleor bone. Accordingly, if the probiotic substance is added tobreakfast-cereals, it may be shaped like cereals. Or, the probioticsubstance may be added to a snack as chips. Additionally, the probioticsubstance may be added with flavors that used to prepare the foodproduct.

In one embodiment, the probiotic substance may be coaled with a moisturebarrier component. In principle, any food-grade substance having waterrepelling or impermeable properties may be selected. Suitable moisturebarriers may be, for example, waxes (paraffin wax, beeswax, carnaubawax, candellila wax, microcrystalline wax, rice bran wax, shellac,lanolin, hydrogenated castor oil, jojoba oil), fatty acids (for example,oleic acid, stearic acid, palmitic acid, lauric acid), monoglycerides,diglycerides and triglycerides (for example, MCT oil, triglyceridesbased on coconut/palm kernel oil), vegetable oils and fats (for example,rapeseed, sesame, cornseed, nut, cottonseed, peanut, sunflower, linseed,olive, soy bean, cocoa butter), hydrogenated or hardened vegetable oilsand fats, oils and fats of animal origin (for example, beef, poultry,pork, for example, beef tallow, lard and fish oil), hydrogenated orhardened oils and fats of animal origin, dairy fats (for example,milkfat, butterfat), proteins (for example, gluten, zein, sodium andcalcium casemate), phospholipids (for example, lecithin), carbohydrates,for example, cellulose and cellulose derivatives (for example,hydroxypropyl methylcellulose, ethylcellulose, methylcellulose,carboxymethyl cellulose), carrageenans, sorbitan esters (for example,mono-oleate, -palmitate, -stearate, trioleate) and mineral oils and fats(for example, paraffin).

The preparation of the probiotic substance after selection of (heprobiotic microorganism and further components of the protecting matrixmay be performed in any suitable way. A few principle steps ofpreparation of the probiotic substance may usually comprise the steps;concentrating the probiotic yield from a fermentor to a solid content ofat least 10% and cfu counts of at least 5×10 cfu/g paste, wet mixing ofthe probiotic concentrate with the other protective components, hydrogelformation in a desirable shape, drying and packaging. If probiotic micromatrix particles are required then a grinding and sieving steps areadded. If additional moisture barrier coating is required then the dryprobiotic substance is coated immediately after the completion of thedrying step.

Most of these steps, for example “wet mixing” and “drying”, may besubdivided, for example “mixing only few of the ingredients, drying themby one drying method, adding other ingredients to the mixture, mixing,and drying again with the same or different drying method.

For example, a wet mix of the micro-organisms and further components, isprepared by mixing all components in water, the slurry is then pouredinto molds of a desired shape and let to harden by adding across-linking chemical or by cooling the slurry, or hardening the gelslurry first and then slicing, chopping or shaping the hydrogel to adesired shape. Then the molded chopped, or shaped hydrogel may be driedto water activity (A_(w)) below 0.2, preferably below 0.1. Possibledrying processes include air dryers or convection ovens, belt dryers,vacuum dryers, fluidized bed dryers, rotary dryers, just to mention afew. Alternatively, all components can be added to the wet probioticconcentrate without the addition of more water. For example, trehaloseis first mixed in a probiotic concentrate (that contains about 10-20%solids), and then egg albumen or soy protein isolate and polysaccharidesare added. Additional amount of proteins may be added to obtain semi-drypowder that can be further granulated, dried and sieved to specific sizerange of probiotic granulated-matrix particles.

The preparation of probiotic substances in a shape of crispy flakes ortreats involves a vacuum drying process where the product temperature isset above the freezing temperature of the probiotic substance. Ingeneral, vacuum drying are performed in two steps. The first stepinvolves moderately reduced pressure (ca. 5000 mTOR) and high shelftemperatures (ca. 5-50° C.), whereas the second step involves lowerpressures (e.g., higher vacuum such as not more than 100 mTOR) whilemaintaining higher shelf temperature (up to about 50° C.).

This process can be achieved using a programmable control system forvacuum pressure and product temperature. The vacuum and temperatureconditions for the first drying step is adjusted empirically accordingto the size of the drier, heat transfer capacity, and the product load,but the goal is to keep the product above its freezing temperature whilemaximizing the water evaporation rate. In one embodiment, thetemperature is initially maintained at about 40° C. for about 6 hours,until most of the free water evaporated from the material, followed bygradually increasing the vacuum from the initial set up of ca. 5000 mTORup to 100 mTOR, then maintaining these drying conditions until the wateractivity of the probiotic substance is sufficiently low (see valuesgiven above). Following this protocol, the drying procedure is completedwithin 24 hours without substantially compromising the probioticviability. The large surface area of the shaped, sliced or choppedhydrogel greatly increases evaporation rate without the need to “boil”or foam the product in thin layers as indicated by other disclosures,thus eliminating inconsistent drying conditions and splattering of thefoamed product solution within the vacuum chamber. Additionally, thedisclosed composition and method of drying enable higher loadingcapacity of product in the vacuum or freeze drier as compared to otherdrying methods of materials with high sugar contents (i.e., foamformation).

The flake or treat may have the shapes as indicated above and be of anysuitable, adequate or desired form. For example, they may have the formof spheres, cubes, pyramids, tablets, long tubes or any complexthree-dimensional form. Furthermore they may have a form thatcorresponds to the food product to which they are added. For example, ifthe treats are added to a pet food for dogs, they may have the form ofbones, animals, cats or other forms that fit with the food product.

In still another embodiment, the dried probiotic substance may be coatedto further protect the micro-organism from deleterious effect ofsubsequent absorption of water during the shelf-life of the foodproduct. The coating may be done by any suitable coating technique, forexample, spraying, melt or solvent coating equipment, fluidized bedcoater, drum coater or pan coater, just to mention a few.

The amount of coating depends on the size and form of the probioticsubstance. Generally, the amount of coating compound is higher forsmaller size particles. Accordingly, the amount of the coating compoundfor probiotic micro-matrix or granulated matrix particles is from 10 to50%, more preferably 20 to 40% of the total weight of the coatedprobiotic particles. The amount of the coating compound for flake ortreat shapes probiotic substance is from 5 to 30%, more preferably 10 to20% of the total weight of the coated probiotic treat. It is alsounderstood that, for the purposes described herein, the coating processcan result in a single layer of one compound or a mixture of compoundsor to multiple layers of one or more compounds.

In a further embodiment, the probiotic substance provides gastricprotection to the probiotic microorganisms. While the sugar component inthe protective matrix immediately dissolves upon intact with the animalstomach juice, the polysaccharide protein matrix retains its form in theacid environment, thereby protecting the embedded microorganisms fromdigestive incursions. The matrix, however, slowly disintegrate in thealkali-phosphate environment of the gut and hence liberating the intactprobiotic microorganisms to colonize the animal gut.

In additional embodiment, the probiotic product may be used exclusivelyas an entire food product, for example as a treat or a supplement, oradded and mixed with a food product, or be used in its powder form tocoat an existing food product, for example for top-coating pelleted orextruded feeds.

The following examples are given by way of illustration only and in noway should be construed as limiting the subject matter of the presentapplication.

EXAMPLES

The subject matter of this disclosure is now described with reference tothe following Examples. These Examples are provided for the purpose ofillustration only, and the subject matter is not limited to theseExamples, but rather encompasses all variations which are evident as aresult of the teaching provided herein.

Example 1 Preparation of Dry and Stable Probiotic Substance

Basic Formulation

300 g of trehalose (Cargill Minneapolis, Minn.) was added to 1000 mlwater and allowed to completely dissolve. Soy protein isolate (100 g,Feam Natural Foods, Mequon, Wis.) and soy hydrolysate (20 g,Sigma-Aldrich, St. Louis, Mo.) were added under vigorous mixing using astandard household blender. Sodium alginate (20 g) was then mixed intothe slurry and allowed to cool down to room temperature. Lactobacillusparacasei (100 g frozen concentrate direct from fermentation harvest)was then added to the slurry under vigorous mixing until a smooth anduniform thick gel was achieved. The composition of the hydrogel isprovided in Table 1.

TABLE 1 Hydrogel composition (g dry weight/100 ml water) Trehalose 30 gSoy protein isolate 10 g Soy protein, hydrolysate  2 g Sodium Alginate 2 g L. paracasei paste 10 g

Production of Probiotic Flakes

Five grams of calcium phosphate dibasic was mixed in the basic formulafollowed by 5 g of gluconolactone and the slurry was allowed to harden(solid hydrogel) at room temperature over the next 4 hours. The firm gelwas sliced to thin and long leafs, through cheese grinder. The thinleafs were loaded on a tray (13×10 inch) and placed in a freeze drier(Virtis Advantage, Virtis, Gardiner, N.Y.). The condenser was set to−70° C. and shelf temperature was set to +40° C. The vacuum was theninitiated and controlled at about 5000 mTOR with an external vacuumcontroller (Thyr-Cont, Electronic, GmbH). The wet product temperaturefell to and stabilized at about −5 to 0° C. The chamber atmosphericpressure was then gradually decreased as the product temperature startedto warm up (measured by a pair of temperature sensors plugged in the wetmaterial), until rail vacuum pressure of 100 mTOR was established. Overthis time period of increasing vacuum, the product temperature wascarefully maintained between −5° C. and +5° C. Twenty four hours afterestablishing full vacuum, the dried product was taken out of the freezedrier. The water activity (A_(w)) of the probiotic substance after thedrying protocol was 0.05 (Measured by HygroPalm Awl, Rotonic Huntington,N.Y.).

Production of Probiotic Treats

The above basic formulation slurry was poured into muffin plates havingmolds of small hearts or stars shapes and allowed to harden (solidhydrogel) at room temperature over the next 4 hours. The platescontaining the molded hydrogel was placed in a freeze drier and allowedto dry as described above.

Production of Probiotic Micro Matrix Particles

The above basic formula was extruded or dripped into a 1000 ml bath(held at 0-5° C.) containing 5 g CaCl₂ and 300 g trehalose using asyringe equipped with 18 G needle. The CaCl₂ bath was gently stirredwhile injecting the slurry. The matrix strings or drops were allowed tocross-link for 30 minutes and then harvested and blotted on paper towel.The strings or drops were first dried in a convection oven at 35 degreeC. until water activity of the material reduced to 0.5 then they weretransferred to a freeze drier for final drying of about 24 hours. Thedry drops or strings (Aw=0.06) then ground to fine powder using standardcoffee grinder and sieved through 50-250 micron screens.

Alternatively, probiotic micromatrix particles can be obtained bygrinding and sieving already dried probiotic flakes or treats asdescribed above.

Production of Probiotic Granulated Matrix Particles

600 g of trehalose (Cargill Minneapolis, Minn.) was added to 1200 ml ofconcentrated paste of Lactobacillus acidophilus (20% solid concentratedirect from fermentation harvest) and allowed to completely dissolve.Soy protein isolate (200 g, Fearn Natural Foods, Mequon, Wis.) and soyhydrolysate (20 g, Sigma-Aldrich, St. Louis, Mo.) were added undervigorous mixing using a standard household mixer. Sodium alginate (40 g)was then mixed into the slurry and allowed to cool down to roomtemperature. After a smooth, thick and uniform gel was achieved, 5 g ofcalcium carbonate was added. Immediately after, 1000 g of egg white(Sigma-Aldrich, St. Louis, Mo.) was slowly added under vigorous mixinguntil a semi-moist free flowing powder was obtained. The granulatedprobiotic powder was dried in a convection oven at 40 degree C. for 2 hfollowed by a vacuum drying for 24 h to obtain dry (Aw<0.06) granulatedmatrix particles. The granulated particles can be sieved to specificsize range through a series of 50-500 micron sieves. The composition ofthe probiotic granulated matrix particles is provided in Table 2.

TABLE 2 Hydrogel composition Trehalose 60 g Soy protein isolate 20 g Soyprotein hydrolysate  2 g Sodium Alginate  4 g L. acidophilus paste 120g  Egg white 100 g 

Recovery of L. Rhamnosus after Production of Dry Micromatrix Particles.

L. rhamnosus micromatrix particles were produced as described above. CFUcounts of concentrated L. rhamnosus after harvesting from a fermentorand centrifuging were 5^(χ)10¹⁰/g paste and solid content was 24%. Aftermixing the probiotic with all protective components, forming hydrogel,chopping the hydrogel to small threads, drying in a freeze-drier for 24h, grinding and sieving to particle size between 50-250 micron, the CFUcounts were 9.4^(χ)10⁹/g dry micromatrix. This represents a loss of 0.28log of probiotic activity during the manufacturing process (asillustrated in FIG. 1).

Stability of L. rhamnosus in 40° C. at 33% Relative Humidity (RH).

L. rhamnosus probiotic micromatrix particles were prepared and dried asdescribed above. The dried probiotic particles were placed intemperature and humidity control incubators set at 40° C. and 33%relative humidity for 4 weeks. Viabilities of bacteria dried in only in10% trehalose solution or in the protective mixture described hereinwere measured on a weekly interval. FIG. 2 shows that the protectivemixture provided a significant protection to that of trehalose alonedried bacteria.

Stability of L. Rhamnosus Micromatrix Particles in Simulated GastricJuices

Micromatrix particles (50-250 micron) containing either L. rhamnosus(LGG), L. acidophilus (LA5) or L. paracase (LPC) were prepared and driedas described above. The micromatrix particles were then exposed for 2hours to simulated gastric juice (full stomach—12% non fat skim milk, 2%glucose, 1% yeast extract and 0.05% cysteine; pH 2; or emptystomach—0.32% pepsin, 0.2% sodium chloride, pH 1.2). Bacterialviabilities were recorded before and after the exposure to the simulatedgastric juices. FIG. 3 demonstrate a significant protection of theprobiotic bacteria in the micromatrix substance in simulated gastricconditions.

Coating of the Probiotic Substances

Probiotic substances were coated with a fat-based moisture barrier (amixture of 20% jojoba oil, 70% cocoa butter and 10% beeswax) in a drumtumbler at a temperature of 40 degrees C. The moisture barrier wassprayed on using a spraying nozzle while the dry material is agitated inthe drum tumbler to ensure homogeneous coating. The total amount ofcoating was about 20% (of the uncoated probiotic substances) forprobiotic flakes and treats, and 40-50% for probiotic micromatrixparticles.

Example 2 Preparation of Probiotic Pet Food

Pet food for dogs that is commercially available was first dried in aconvection oven to a water activity of 0.1, and then coated withprobiotic micromatrix particles in a drum tumbler. The pellets werefirst sprayed with about 5% of fat-based moisture barrier (a mixture of40% chicken fat, 40% cocoa butter and 20% beeswax), then mixed with themicro matrix particles (usually 0.1-0.5% of total pet food that providesa dosage of 10⁸ CFU/g) and finally sprayed with additional coat of thefat-based moisture barrier. The total amount of coating was about 15%(of the pet food). Coating time was about 30 minutes.

Example 3 Preparation of Fish Feed with Several ProbioticMicro-Organisms

Pelleted feed for fish was prepared with a mixture of severalprobiotics. Probiotic micromatrix particles containing a mixture of L.rhamnosus, L. acidophilus and Bifidobacterium lactis (DSM 20215) wereprepared as described in Example 1. Fish feed that is commerciallyavailable was first dried in a convection oven to a water activity of0.1, and then coaled with probiotics micromatrix particles in a drumtumbler. The pellets were first sprayed with about 5% of fat-basedmoisture barrier (a mixture of 40% fish oil, 40% cocoa butter and 20%beeswax), then mixed with the micro matrix particles (usually 0.1-0.5%of total fish feed that provides a dosage of 10⁷ cfu/g) and finallysprayed with additional coat of the fat-based moisture barrier. Thetotal amount of coating was about 10% (of the fish feed).

Example 4 Preparation of Probiotic “Chips” (as Shown in FIG. 4)

Probiotic chips were prepared with a mixture of L. rhamnosus and L.acidophilus as described in Example 1. The basic formula was added withabout 1-2% of probiotic pastes instead of the standard 10-20% load. Fivegram of calcium phosphate dibasic was mixed in the basic formulafollowed by 5 g of gluconolactone and the slurry was pored into longplastic tubes of 1-inch diameter and allowed to harden (solid hydrogel)at room temperature over the next 4 hours. The firm “hot-dog” shaped gelwas sliced to thin discs, loaded on a tray (13×10 inch) and placed in afreeze drier and a drying procedure applied as described in Example#1.The dried probiotic chips product was taken out of the freeze drier andpacked under nitrogen in small aluminum foiled packs that provide tastyand crispy treat containing an effective dose of 10⁸ CFU/per serving.FIG. 4 shows an example of probiotic chips containing 10⁸ CFU/serving ofrhamnosus produced as described herein.

Example 5 Preparation of Probiotic Cereals

Breakfast cereal that is commercially available was first dried in aconvection oven to a water activity of 0.1. Probiotic chips containing astandard 10-20% load of probiotics were produced and further coated witha moisture barrier as described in Examples 1 and 4. The cereals werethen mixed with 0.1-0.5% probiotic chips (to provide a dosage of 10⁸cfu/g).

Example 6 Alternative Delivery Forms of Probiotic Matrix Substances inthe Lid of a Container or a Bottle of Beverage Drink

In general, all forms of the probiotic substances produced as in Example1 or Example 4 work similarly well, confirming the high versatility ofusage and application of the materials. The probiotic substancesproduced as described herein can be provided in small bags or paper“stick” packaging or in any other manner along with an edible foodproduct. The dry micromatrix particles or chips may also be provided inthe lid of a yogurt container or a bottle of beverage drink to be mixedwith the liquid food product for consumption.

REFERENCES

The following references are referred to herein.

-   Bronshtein, V. (WO2005117962). Preservation by vaporization (2005).-   Cavadini, C, Ballevre, O. and Gaier, H. (EP 0 862 863). Cereal    product containing probiotics.-   Crowe, J. H., Carpenter, J. F. and Crowe, L. M. (1998). “The role of    vitrification in anhydrobiosis. Review.” Arum Rev Physiol. 60:    73-103.-   Farber, M. and Farber, J. (WO 03/088755). Delivery systems for    functional ingredients (2003).-   Giffard, C J. and Kendall, P. (US 2005/0079244). Foodstuff (2005).-   Hughes, V X. and Hillier, S. L. (1990). “Microbiologic    characteristics of Lactobacillus products used for colonization of    the vagina.” Obstet Gynecol. 75: 244-248.-   Kenneth, A.M. and Liegh, B. T. (U.S. Pat. No. 6,900,173).    Perioperative multivitamin protein bar for use in preparing an    individual for fast surgical recovery (2005).-   Ko, S. T. and Ping, Y. A. T. (WO 02/058735). Methods of preparing    biological materials and preparation produced using the same.-   McGrath, S. and Mchale, A. P. (EP 1382241). Storage and delivery of    microorganisms (2004).-   Porubcan, R. S. (US 2004/0175389). Formulations to increase in vivo    survival of probiotic bacteria and extend their shelf-life (2004).-   Shah, N. P. (2000). “Probiotic bacteria: selective enumeration and    survival in dairy foods.” Journal of Dairy Science 83: 894-907.-   Ubbink, J. B., Zammaretti, P. S. and Cavadini, C. (US 2005/0153018).    Probiotic delivery system (2005).

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this subject matter has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations can bedevised by others skilled in the art without departing from the truespirit and scope of the subject matter described herein. The appendedclaims include all such embodiments and equivalent variations.

1. A probiotic substance comprising viable probiotic bacteria preservedin a dried matrix of at least one sugar, at least one protein, and atleast one polysaccharide.
 2. A probiotic substance according to claim 1,wherein the sugar in the dried matrix is a disaccharide.
 3. A probioticsubstance according to claim 2, wherein the disaccharide is selectedfrom the group consisting of sucrose and trehalose.
 4. A probioticsubstance according to claim 1, wherein the protein in the dried matrixis selected from the group consisting of egg white, soy protein isolate,soy hydrolysate or a combination thereof.
 5. A probiotic substanceaccording to claim 1, wherein the polysaccharide is a cross-linkablegel, most preferably selected from the group consisting of a combinationof alginates with different viscosities, agarose, pectin, and chitosan.6. A probiotic substance according to claim 1, wherein the dried matrixis comprised of 20% w/v to 60% w/v trehalose, 2% w/v to 20% w/v eggwhite or soy protein isolate and 0.5% w/v to 10% w/v alginates.
 7. Aprobiotic substance according to claim 1, wherein the probioticmicroorganism comprises 10⁵ to 10¹⁴ viable micro-organisms per gram ofdry substance.
 8. A probiotic substance according to claim 1, whereinthe probiotic substance is coated with additional moisture barrier.
 9. Amoisture barrier according to claim 8, wherein the barrier comprises anyfood-grade substance having water repelling or impermeable properties.10. A moisture barrier according to claim 8, wherein the barriercomprises a mixture of oil based substances.
 11. A probiotic substanceaccording to claim 1 wherein the probiotic microorganism retain most ofits initial biological activity after manufacturing, after extendedexposure to high temperature and humid conditions and after gastricexposure.
 12. A human food product comprising one or several probioticsubstances comprising viable probiotic bacteria preserved in a driedmatrix comprising at least one sugar, at least one protein, and at leastone polysaccharide.
 13. A human food product of claim 12, wherein thefood product is in a moist, semi-moist, or semi-dry form.
 14. A humanfood product of claim 12, wherein the food product is in a powdered,particulate, pellet, tablet, capsule, colloidal suspension or liquidform.
 15. A human food product of claim 12, wherein the food product isadded to a bar, liquid formula, or cereal or another food product.
 16. Ahuman food product of claim 12, wherein the food product is in the formof a treat, a nutraceutical food additive or a pharmaceutical foodadditive.
 17. An animal feed product comprising one or several probioticsubstances comprising viable probiotic bacteria preserved in a driedmatrix comprising at least one sugar, at least one protein, and at leastone polysaccharide.
 18. An animal feed product of claim 17, wherein thefeed product is in a moist, semi-moist, or semi-dry form.
 19. An animalfeed product of claim 17, wherein the feed product is in powdered,particulate, pellet, tablet, capsule, colloidal suspension or liquidform.
 20. An animal feed product of claim 17, wherein the feed productis added to a bar, liquid formula, or cereal or another food product.21. An animal feed product of claim 17, wherein the feed product is inthe form of a treat, nutraceutical food additive or a pharmaceuticalfeed additive.
 22. An animal feed product of claim 17, wherein theanimal is chosen from terrestrial or aquatic animals.
 23. A method ofmaking probiotic food or feed substance comprising: a) mixing apreparation of probiotic micro-organisms with at lease one sugar, atleast one protein, and at least one polysaccharide to obtain a smoothand uniform gel in a desired shape and form; and b) vacuum drying thepreformed solid gel to a water activity below 0.1.